Biosensors, which can be subdivided into affinity based and metabolic biosensors, serve for the preferably selective and specific detection, respectively for concentration measurement, of a predetermined analyte contained in a liquid sample to be examined. The large group of heterogeneous biosensors includes, in such case: a receptor layer, which can have a large number of receptors immobilized on a surface, e.g. receptors such as antibodies, haptens, DNA, cells or enzymes; a signal transducer, which outputs a measurement signal correlated with the amount of the analyte bound on the receptor layer; and a signal processing system for amplification and/or additional processing of the measurement signal output by the signal transducer.
The analyte can be a substance to be detected in a liquid sample, for example, a substance such as a protein, a peptide, an antibody, an enzyme or some other substance to be detected in biochemical, biological or medicinal systems. The analyte can, however, also be cells, cell components or DNA or, for example, active ingredients, which are to be detected or determined in the context of environmental monitoring or in the monitoring of water quality.
In the case of affinity based biosensors, it can, in given cases, be necessary to connect to the receptors of the receptor layer, besides the analyte, yet other substances. A substance, which preferably binds with high specificity and selectivity to a receptor, is here generally referred to as the target molecule or also as a ligand for such receptor. Thus, in the case of a given biosensor, the analyte represents a target molecule for the receptors. Other target molecules can, however, also be molecules different from the actual analyte, and these likewise attach to the receptors of the receptor layer, and compete, for example, with the analyte, for binding locations of the receptor layer.
In the case of metabolic biosensors, there is catalytic conversion of the analyte and/or other target molecules after their temporary binding to the receptor layer, which, in the case of metabolic biosensors, contains at least one enzyme, a cell, a cell component or other catalytically effective biomolecule, which can catalytically convert the analyte, in given cases, with additionally present substances.
For detection or for determining concentration of an analyte in a liquid sample, the liquid sample is brought in contact with the receptor layer, so that analyte molecules and, in given cases, other target molecules present in the liquid sample attach to the receptor layer or are converted by the receptor layer. In this way, physical and/or chemical changes are effected, for example, a change of coating thickness, index of refraction, light absorption or electrical charge. These changes can be detected and quantitatively determined by means of the signal transducer. Suitable signal transducers for registering said changes include, for example, optoelectric sensors, and amperometric or potentiometric sensors. The target molecules bound on the receptor layer can also be marked directly or indirectly with a marker, which has, for example, luminescent or magnetic properties, or be marked subsequently directly or indirectly after the binding of the target molecules on the receptor layer. In this case, an optical sensor is suitable as signal transducer for registering the luminescence, or a magnetic sensor for registering magnetic properties. Further suited as markers are also enzymes, which catalyze a following chemical reaction, wherein the course of the reaction can be registered with correspondingly suitable signal transducers.
Numerous different heterogeneous biosensor methods are known. The most frequently used methods will be mentioned and briefly explained by way of example as follows:                Direct method: The analyte binds on receptors of the receptor layer, and no other target molecules are present. In a modification of the method, the analyte is reacted with one or more additional substances (markers) before or after the binding on the receptors, this serving, as a rule, for introducing, directly or indirectly, a physical property, which the analyte does not possess, and so make the binding of the analyte detectable by determining this provided physical property. In the case of the direct method, thus, the binding of the analyte on the receptor layer is directly detected or detected indirectly by detecting the marker reacted with the analyte.        Competitive method: Added to the liquid sample to be examined for an analyte content is, as a rule, a further target molecule (competitor) in known concentration or activity and the liquid to be measured so obtained is then applied to the receptor layer. In such case, there is competition between analyte and competitor for binding on the receptors of the receptor layer. The competitor possesses, as a rule, a marker, which, in turn, introduces, directly or indirectly into the system, a physical property, which can be detected. In the case of the competitive method, thus, the binding of the competitor on the receptor layer is directly or indirectly detected by detecting the competitor marker. The original analyte concentration of the liquid sample can then be derived from the measurement signal.        Binding inhibition test: Added to the liquid sample to be examined for an analyte content is a complementary molecule in known concentration or activity, which bonds to the analyte. Subsequently, the so formed, measured liquid is led over the receptor layer, which has receptors, which bind the free complementary molecules, not bonded to the analyte. This binding can be detected directly by means of suitable methods. Alternatively, the complementary molecules can be reacted directly or indirectly with a marker before or after the binding on the receptors or before or after the bonding to the analyte. In the case of a binding inhibition test, thus the binding of the complementary molecules on the receptors is detected directly or the markers on the complementary molecules are detected directly or indirectly. Then, the analyte concentration of the liquid sample can be derived from the measurement signal.        
For preparing the receptor layer, the receptors are immobilized on solid carrier materials, i.e. bound to these. This can occur, for example, by unspecific adsorption of the receptors on the surface of the carrier material, the so called substrate. However, also covalent bonding of the receptors or bonding via a number of other bonding layers are established methods for preparation of receptor layers. Thus, for example, streptavidin can be bonded covalently on a solid carrier surface and, for preparing the receptor layer, biotin-conjugated antibodies connected on the streptavidin binding layer using the affinity interaction between biotin and streptavidin. In general, one speaks of the total layer system constructed on the surface of a solid carrier material as the ‘sensor matrix’ of the biosensor, wherein the last layer is the receptor layer.
The sensor matrix with the receptor layer is, in many applications, applied on a biochip integrated in a microfluidics system. These biochips are, as a rule, single-use products, which are discarded, respectively replaced by a new biochip, after a single performed determination or concentration measurement of the analyte. Also, in the case of metabolic biosensors, the chip, or the cartridge, which contains the sensor matrix, must be replaced at regular intervals. The reason for this is, generally, the low stability and robustness of biological receptors as well as the often high affinity, with which target molecules are bound on the receptors. Thus, coupled with conditions, under which target molecules bound on the receptors are shed from the receptors is, as a rule, also a more or less large destruction of the receptor layer. Known from the relevant technical literature are only few special cases, in which there is described such a regeneration of the receptor layer, i.e. the almost complete removal of the target molecules while retaining the essentially unchanged functionality of the receptor layer. According to the state of the art, this is, however, not possible for the large majority of biosensors, especially for biosensors for determining proteins.
Automated or semi-automated bioanalyzers, in which single-use chips carrying a receptor layer are used, are known. Thus in European Patent EP 1 343 011 B1, for example, an apparatus is described for electrochemical detection of a nucleotide sequence. This apparatus has a replaceable analytical cassette, preferably embodied as a single use-component, for introducing the liquid sample. The cassette has a measuring electrode with a receptor layer, on which the nucleotide sequence selectively and specifically binds. The analytical cassette is connectable with a calculation system via an analog interface, wherein an electrochemical detection of the nucleotide sequence is performable by means of the calculation system.
For application of biosensors, especially of affinity based biosensors, in process measurements technology, for example, for automated monitoring of a biotechnological process in an industrial plant or for automated monitoring of water, for example, for residues of medicines or for the content of endocrinally active substances, it is, in contrast, desirable, to be able to perform a plurality of measurements one after the other, without requiring for each measurement a replacement or an exchanging of a chip or other component containing the sensor matrix. A possible application of biosensors for such measuring tasks can be a bioanalyzer, which is fed, especially online, liquid samples of a process medium to be monitored. Optionally, the liquid samples can be pretreated, preferably automatically, with reagents, for example, with other target molecules, markers, etc. for performing the above described methods. The bioanalyzer includes furthermore: a receptor layer, to which the liquid sample, or the liquid to be measured, as obtained by pretreating, is fed; a signal transducer, which outputs a measurement signal correlated with the number of analyte, or target, molecules bound on the receptor layer; and a signal processing system, which further processes the measurement signal and outputs a measured value, especially one derived from the measurement signal. The supplying of the samples and, in given cases, reagents to the receptor layer and the draining of the liquid sample, or liquid to be measured, after transpired measuring, can be performed in an automated or semi-automated analyzer by means of pump apparatuses, pneumatic systems or other liquid transport systems. In order to keep the required reagent amount and therewith, also the liquid volume to be disposed after transpired analysis, small, supply and drain lines for the sample and, in given cases, reagents should have cross sections, which are as small as possible, e.g. in the sub-mm range. Such liquid lines with cross sections in the sub-mm range are referred to in the following also as microfluidic ducts. A module of a bioanalyzer, which has such microfluidic ducts, is also referred to as a microfluidic unit.
In order to minimize the maintenance effort for a bioanalyzer used for such purposes, or in order to increase the degree of automation, there is a need for biosensors suitable for performing a plurality of measurements one after the other, wherein the sensor matrix, respectively its receptor layer, is essentially completely regeneratable, or renewable, in situ, with high reproducibility.
Known from the literature are different approaches concerning how sensor matrices or receptor layers of biosensors can be regenerated. Frequently, in such case, the solvent is altered, in order to bring about a releasing of the binding between receptor and target molecule. Frequently for this, the pH-value of the solution is changed. Also, changes of ionic strength or solvent polarity or the addition of specifically acting substances are often applied, in order to achieve a regeneration of receptor layers. Disadvantageous in these methods is, however, that, in order to enable a multiple regeneration of the receptor layer, for each receptor-target molecule interaction, the optimal compromise between optimum regeneration efficiency and minimum destruction of the receptors must be ascertained in complex test runs. A renewing of the receptors, or the receptor layer, is not possible according to these methods.
A. T. Tüdös, E. R. Lucas-van-den Bos, E. C. A. Stigter, “Rapid surface plasmon resonance-based inhibition assay of deoxynivalenol”, Journal of Agricultural and Food Chemistry 51 (2003), 5843-5848, describe such a regeneration method for a receptor layer with haptens as receptors that are especially small and robust. Between 499 and 717 regeneration cycles are claimed. For an immunoassay, in the case of which the receptors are proteins, no receptor layers are known, which can be multiply regenerated with similar success.
Known from European Patent EP 2 189 793 A1 is that, for removing the target molecules from the receptors of the respective receptor layer, enzymes can be applied, which selectively decompose the target molecules connected to the receptors. In such case, it must be heeded, however, that the used enzymes do not equally attack the receptors, and this strongly constrains the ability to apply this technique. Due to the chemical nature of such enzymatic decomposition of the bound target molecules, maintaining the same receptor density following multiple performances of the method appears extremely improbable.
A further approach for regeneration of the receptor layer of a biosensor involves binding the receptors via oligonucleotides, thus short DNA, or RNA, molecules. For this, oligonucleotides are covalently bonded to a surface and the receptors or molecules, which form a corresponding binding layer for binding receptors, are conjugated with the complementary oligonucleotide. Specific affine interaction between the complementary oligonucleotides leads to an immobilizing of the receptors on the surface and therewith to the forming of a receptor layer. The affine interaction between the complementary oligonucleotides can be removed chemically or by increasing the temperature, so that the receptors are released from the surface. However, this method in the presence of proteins, e.g. in the presence of antibodies as receptors, is generally not performable, since proteins, as a rule, denature when faced with temperature increase to over 40° C., or in the presence of the reagents needed for the release. In such case, it can come, especially in the case of high protein content, in the case of denaturing the proteins, to the forming of difficultly soluble, protein aggregates, which can only be removed incompletely without aggressive chemical cleaning and which, by the agglutinating and accreting, for example, of liquid lines of the liquid supply- and -drain system, can affect extremely disturbingly the functionality of an automated or semi-automated bioanalyzer. The same is true when the molecules bound on the receptor layer are bridged or cross linked with one another. Also in this case, aggressive chemical conditions must be applied for releasing these bridged or cross linked molecules. In the face of the aggressive chemical conditions, which would be required for removing the protein aggregates, moreover, also the surface bonded oligonucleotides are no longer stable.
Further known from the literature are first approaches for an electrochemical removal of simply structured, biological layers. In contrast to the chemical removal of the sensor matrix, respectively receptor layer, with purely chemical means, e.g. by acids, alkaline solutions or solvents, in the case of the electrochemical removal of the receptor layer, an electrical current flow via the substrate carrying the receptor layer is effected, which leads to oxidative or reductive release (desorption) of the receptor layer, respectively sensor matrix.
Thus, for example, in the articles Seokheun Choi, Junseok Chae “Reusable biosensors via in situ electrochemical surface regeneration in microfluidic applications”, Biosensors and Bioelectronics 25 (2009) 527-531, Seokheun Choi, Junseok Chae, “A regenerative biosensing surface in microfluidics using electrochemical desorption of short-chain self-assembled monolayer”, Microfluidics and Nanofluidics 7 (2009) 819-827 or Christie A. Canaria et al., “Formation and removal of alkyithiolate self-assembled monolayers on gold in aqueous solutions” Lab on a Chip 6 (2006) 289-295, approaches are described for applying receptors on gold surfaces by means of alkyl thiols as binding molecules, in order, in this way, to provide a receptor layer. Also described in these articles are investigations concerning the problem of how to remove the alkyl thiols used as binding molecules. Problems occur, in such case, especially through re-adsorption of already released alkyl thiols and hydrogen evolution occurring from electrochemical decomposition of the alkyl thiols. Additionally, there is in the case of repeated preparation and removal of the layers a continuous drift observed in the surface occupation, which indicates non-complete removal. In none of the cited article is it explored, whether the cleaning effectiveness is equal in the case of different occupations of the receptor layer. This is, however, indispensable for application in an automated bioanalyzer for process measurements technology, since biosensor measurements are performed, as a rule, based on one or more reference measurements, in the case of which the occupation of the receptor layer occurs, as a rule, either not or almost completely. Especially in the case of SPR measurements, as described in the articles, the quality of the reference measurement is decisively important for the quality of the measurement results. Thus, for example, in the case of Seokheun Choi, Junseok Chae “Reusable biosensors via in situ electrochemical surface regeneration in microfluidic applications”, Biosensors and Bioelectronics 25 (2009) 527-531, it is not to be assumed that the cleaning effectiveness is the same in the case of receptor layers, on which much fibrinogen is bound (FIG. 3c), as in the case of receptor layers, on which only little fibrinogen is bound.
For the automated or semi-automated application of the biosensors in process measurements technology, it is important that sequentially executed concentration determinations deliver measurement results comparable with one another. Therefore, a system is needed, which delivers even after a plurality of regenerations, or renewals, of the receptor layer, for example, after 50, 100 or more regeneration- or renewal cycles, for equal target molecule concentrations, essentially equal measurement signals, i.e. measurement signals of comparable intensity, especially in the case of reference of the measurement signals to the reference measurement.